Biogeneration of silver nanoparticles from Cuphea procumbens for biomedical and environmental applications

Nanotechnology is one of the most important and relevant disciplines today due to the specific electrical, optical, magnetic, chemical, mechanical and biomedical properties of nanoparticles. In the present study we demonstrate the efficacy of Cuphea procumbens to biogenerate silver nanoparticles (AgNPs) with antibacterial and antitumor activity. These nanoparticles were synthesized using the aqueous extract of C. procumbens as reducing agent and silver nitrate as oxidizing agent. The Transmission Electron Microscopy demonstrated that the biogenic AgNPs were predominantly quasi-spherical with an average particle size of 23.45 nm. The surface plasmonic resonance was analyzed by ultraviolet visible spectroscopy (UV–Vis) observing a maximum absorption band at 441 nm and Infrared Spectroscopy (FT IR) was used in order to structurally identify the functional groups of some compounds involved in the formation of nanoparticles. The AgNPs demonstrated to have antibacterial activity against the pathogenic bacteria Escherichia coli and Staphylococcus aureus, identifying the maximum zone of inhibition at the concentration of 0.225 and 0.158 µg/mL respectively. Moreover, compared to the extract, AgNPs exhibited better antitumor activity and higher therapeutic index (TI) against several tumor cell lines such as human breast carcinoma MCF-7 (IC50 of 2.56 µg/mL, TI of 27.65 µg/mL), MDA-MB-468 (IC50 of 2.25 µg/mL, TI of 31.53 µg/mL), human colon carcinoma HCT-116 (IC50 of 1.38 µg/mL, TI of 51.07 µg/mL) and melanoma A-375 (IC50 of 6.51 µg/mL, TI of 10.89 µg/mL). This fact is of great since it will reduce the side effects derived from the treatment. In addition, AgNPs revealed to have a photocatalytic activity of the dyes congo red (10–3 M) in 5 min and malachite green (10–3 M) in 7 min. Additionally, the degradation percentages were obtained, which were 86.61% for congo red and 82.11% for malachite green. Overall, our results demonstrated for the first time that C. procumbens biogenerated nanoparticles are excellent candidates for several biomedical and environmental applications.


Results
In the process of nanoparticle synthesis one of the characteristic effects is the color change from light brown to dark brown, which suggests the formation of AgNPs through extracellular activity 41 . Figure 1 shows UV spectral analysis at different time intervals. The UV-vis spectrum of the silver nanoparticles shows the monitoring of the formation of nanoparticles in a period of 6 h, recording the activity every 20 min. The UV-Vis spectrum indicated the presence of a strong and broad band in a range from 350 to 450 nm, and specifically showing a maximum absorption peak at 441 nm, which is attributed to the Surface Plasmon Resonance (SPR) 42 . This is due to the nucleation and growth of nanoparticles produced as a result of the reduction of silver ions present in the solution 43 .
The aqueous extract of C. procumbens has bioorganic compounds bound to the surface of the AgNPs which were identified by FT-IR spectroscopy (Fig. 2) The strong absorption peaks of the leaf extract of C. procumbens were observed at 1500, 1450 and 1100 cm −1 . The change in vibration around 1450 cm −1 of C. procumbens suggested the presence of aliphatic -CH and aromatic-OH groups such as hydroxy flavones and hydroxy xanthones 39 . The vibration around 1100 cm −1 was attributed to the primary amine C-N stretching vibrations of aliphatic amines, the presence of N-H stretching of amine groups, aliphatic C-H stretchings 44 , C-N stretching of amines/ proteins and C-H stretchings, respectively and according to other authors.
The FTIR spectra of AgNPs also showed peaks at 3250, 1550, 1384, 1100 cm −1 due to O-H stretching of polyphenols , C=C or C=O stretch of carboxylic acids 14 , amide stretching, C-O stretching, C-N stretching of amine proteins 27 , alkene C=C stretchings, =C-H bending and bending vibration of C-H stretching 18 , respectively.
The peaks at 1550 and 1450 cm −1 are characteristic of primary amide stretches 45 . These functional groups are representative of the amide or polyphenol groups of C. procumbens, which are responsible for the formation and stabilization of AgNPs.
In the TEM micrographs we can observe that through a high-resolution analysis (HRTEM) in Fig. 3a, the crystalline structure of the AgNPs was corroborated with the measurements of the interplanar distance of www.nature.com/scientificreports/ predominantly nearly spherical. Selected area electron diffraction (SAED) (Fig. 3c), indicated that the measurements of the family of crystalline planes correspond to the metal Ag and to the FCC (Face Centered Cubic) crystalline structure, which we corroborate from JCPDS card 00-004-0783. The size distribution histogram of AgNPs ranged from ∼2-24 nm ( X = 12.35 nm, σ = 2.56 nm, Fig. 3d).
Regarding the characterization of the size, it was found by DLS (dynamic light scattering) that the hydrodynamic size is 23.45 nm, while the polydispersity index (PDI) was 0.242.
The results of the "broth microdilution" test are shown in Fig. 4, to evaluate the antibacterial activity of the AgNPs, the pathogenic strains of E. coli and S. aureus were used. AgNPs were effective against both strains. In E. coli. the maximum zone of inhibition was at a concentration of 0.225 µg/ml. while for S. aureus it was 0.158 µg/ mL, as shown in Table 1.
The percentage of viability decreased with increasing concentrations of the plant extract (Fig. 5a). The IC 50 of the extract are higher than IC 50 of AgNPs ( Table 2). As expected the AgNPs potentiated the effect (Fig. 5b) as it has been observed by other research groups 46 . Likewise, the therapeutic index values indicated that the effect of the AgNPs is higher than the extract alone (Table 3).  www.nature.com/scientificreports/ The ability of the AgNPs to degrade congo red and malachite green dye of the synthesized AgNPs is presented in Fig. 6. Congo red and malachite green are non-biodegradable and toxic azo dyes that can be degraded through the use of NPs. The degradation reaction of congo red and malachite green was monitored by UV-Vis spectrophotometer. Congo red in water showed a band at 496 nm and an electron transition of 343 nm associated with the azo group. While the malachite green degradation reaction showed an SPR band at 496 nm and an electron transition of 351 nm. From these graphs, it is observed that the absorption peak of the dye molecules  www.nature.com/scientificreports/ gradually decreased over time. The absorption peak disappeared and the color of the solution changed from red to colorless. Regarding the degradation percentages, we observed a value of 86.61% for congo red and 82.11% for malachite green, observing a significant decrease in dye degradation from the first contact (Fig. 7).   www.nature.com/scientificreports/

Discussion
According to the potential the AgNPs can be used in three main application including degradation of dyes, antibacterial tests and antitumor agents. The formation of AgNPs was monitored 47 , observing a change in color of the reaction mixture that began to change from a light yellow to dark brown after 6 h, which indicated the generation of AgNPs, due to the participation of a redox reaction of silver metal Ag + ions in silver Ag° nanoparticles through the active molecules present in the extract of C. procumbens48. This color is attributed to the excitation of SPR49. A characteristic and well-defined SPR band for silver nanoparticles was obtained at around λ 441 nm 7 . FTIR measurements of biosynthesized AgNPs were performed to identify possible biomolecules responsible for the stabilization of AgNPs. Previous studies have revealed that carbonyl, amide, and amino groups show a tendency to bond with metal particles. This helps to form a layer on the metallic nanoparticles, which ensures their stabilization and agglomeration. The amide and other functional groups in the extract can probably influence the interaction of AgNPs with peptides or carbohydrates, thus stabilizing them. The smaller size and crystal structure of AgNPs have excellent antimicrobial potential 50 . TEM analysis of the particles provides information on size and formation. The mean sizes of polydispersed silver AgNPs have been shown to be 20 nm. TEM images of silver nanoparticles have shown that the morphology of silver nanoparticles was predominantly quasi-spherical with a mean diameter of 12.3 nm and a standard deviation 2.56 nm. These results are similar to those obtained by other authors using the same methodology [51][52][53] . This is due to the bioreduction from various compounds in the medium, as commonly occurs in green synthesis. In addition, it is possible to corroborate the crystal structure of the AgNPs, in fact it coincided with what is specified by card 00-004-0783 54 with an analysis corresponding to the micrographs of HRTEM and SAED, with this analysis it is determined that AgNPs are indeed composed of the element Ag (FCC). The data obtained from the DLS measurements of hydrodynamic size and polydispersity index (PDI) were 23.45 nm and 0.242 respectively. As can be seen there is a difference of 11.15 nm ± 2.56, this is due to that only the nanoparticle count was made through a small part of the copper Table 2. Antiproliferative activities (IC 50 (µg/mL)) of AgNPs and C. procumbens extracts in cancer cell lines and macrophages. All the experiments were performed in triplicate.   (Fig. 4). AgNPs activity is highly dependent on AgNPs concentration. This is in accordance with what was reported in a previous work 55 . The interface between bacteria and AgNPs can generally be described by the following approaches: first, nanoparticles possess an extremely large surface area that provides better contact and interaction with bacterial cells 18 . Second, their interactions may be between positively charged membrane proteins present on the surface of bacteria and negatively charged AgNPs 56 . Furthermore, the attraction of these NPs to the surface of the cell membrane depends on the particle's surface area. The smaller size of the AgNPs will offer a better surface area that can interact and penetrate the membrane of the cell surface. This will provide a significant bactericidal effect and cause bacterial cell death 56 .
Likewise, it is possible to observe a very important principle; apparently our generated nanoparticles, unlike the extract, exert their antiproliferative effect on tumor cells with much lower concentrations that go unnoticed by macrophages. This fact is of great importance as it could prevent many of the side effects of current cancer treatments. It may be due to that AgNPs are going to sites with greater energetic activity, such as tumors or cancer cells 57 .
It is important to recognize that the biogenerated AgNPs have a powerful antitumor effect on cell lines from various tissues, highlighting its effect on the colon cancer line HCT-116 (IC 50 59 . Other studies also showed the IC 50 of d Fumaria parviflora extract and AgNPs-d Fumaria parviflora against MDA-MB-468 cancer cells were observed at 100 µg/mL and 80 µg/mL respectively 60 . Therefore, it is assumed that the potentiated effect of the nanoparticles is due to the component of the C. procumbens extracts ( Table 2) that is working as a reducing and stabilizing agent of the AgNPs. We observed that the concentration of the extract used for the biogeneration of nanoparticles is sufficient to generate antitumor activity, bioreduce nanoparticles and stabilize them. It should be noted that these results depend greatly on the concentration of metabolites present in the extracts and on the concentrations of the precursor salt used 61 . If we compare with recent studies of AgNPs synthesized by chemical methods, such as the nanoparticles synthesized by Al-Khedhairy, AA and Wahab, R. 62 in which they report an IC 50 value of 9.85 µg/mL, as we can see the IC 50 values are more lower than even the values provided by a chemical synthesis. That makes our research generate greater interest. We attribute this important property to the potentiation of the effect of the AgNPs and the aqueous extract of C. procumbens as a whole.
The importance of our work relies in the therapeutic index (TI) obtained, since its value was high for all tumor cell lines treated with AgNPs compared to the extract ( Table 3). The best TI was achieved with HCT-116 and MDA-MB-468 (TI = 51.07 and 31.53 respectively). In the specific case of the MCF-7 cell line, a value of 27.65 was obtained. and it was compared with Melia dubia 63 and Cassia fistula 64 , which present a therapeutic index of 16.02 and 9.23 respectively,. Additionally, human lung epithelial carcinoma cells (A549) and human breast epithelial cells (HBL100) have been studied through the therapeutic index with silver nanoparticles obtained with plant extracts, obtaining a therapeutic index of 2. 63 . In human cervical cancer (HeLa) cells, metal nanoparticles generated from green synthesis have had therapeutic indices of ≤ 2.5 65 . In addition, we have seen that a similar concentration of the extract and the AgNPs induces inhibition of 50% of macrophage proliferation. Hence, we suspect that tumor cells are less susceptible to damage from flavonoids present in the aqueous extract of C. procumbens, while AgNPs enhance its antitumor effect. This is due to the rapid proliferation of cancer cells, since they require high levels of energy, so they can be more sensitive and have important physiological changes 66 .
Congo red is a diazo-anionic dye 67 . This colorant, in addition to affecting the aesthetics, the transparency of the water and the solubility of oxygen in the bodies of water, has been reported as highly toxic for living beings because it causes carcinogenesis, mutagenesis, teratogenesis, respiratory damage, allergies and problems during pregnancy 68 . The congo red or salt of 3,3'-(4,4'-biphenylenebis (azo) bis (4-amino) disodium naphthylene sulfonic acid is prepared by a tetradiazotization with benzidine and naphthylsulfonic acid. The covalent bonds in the molecule confer stability, which together with the complex molecular structure they make biodegradation and photodegradation difficult. Congo red in an aqueous solution (distilled water) shows a Surface Plasmon Resonance (SPR) band at 496 nm (π → π) and the electronic transition at 351 nm (north → π) is associated with the azo group. In Fig. 6a it is observed that the absorption peak of the dye molecules gradually decreases in a time dependent manner. The solution fades from red to colorless. Malachite green is a cationic triphenylmethane dye that is widely used in various fields as a parasiticide. The catalytic degradation of the malachite green organic dye was controlled by the change in the absorbance of ultraviolet light. In addition, the degradation capacity in liquid medium was evaluated (Fig. 6b) 69 . According to the literature, the photocatalytic activity 33 of NPs depends on the shape, size and crystalline structure of particles 69 . To our knowledge, this is the first time that biosintethized AgNPs are able to degrade this colorant at a low concentration. Our results are really very promising, compared to other authors such as Lateef and Akande 70 , who only reach 80% degradation with 20 µg/mL 70 , which is interesting because the concentration of nanoparticles used in our study is much lower. Even other authors report that using NaBH 4 manage to degrade colorants in a longer period of time 71  www.nature.com/scientificreports/ important to highlight that we are not using any type of additional catalyst 72 , nor radiation or exposure to light, in addition to applying other techniques 73 .

Methods
Biosynthesis. Preparation of the extract of C. procumbens. For the elaboration of the aqueous extract, leaves of C. procumbens were collected in the month of May in the municipality of Villa Victoria, State of Mexico, Mexico, handling of plants were carried out in accordance with Mexican guidelines and regulations, washed thoroughly with distilled water, allowed to dry at room temperature for 15 days, chopped finely and ground, in 100 mL of sterile distilled water, 0.5 g of the treated leaves of C. procumbens were added and heated to boiling point for 5 min. Then was filtered the extract through Whatman No. 1 filter paper (size of 25 µm pore). A second filtration step was carried out using Amicon Ultra-15 30 kDa tubes. To purify the aqueous extract, we performed a second filtration step using an Amicon 30 kDa ultrafiltration unit. The ultrafiltration units were centrifuged at 300 rpm for 10 min.
Synthesis of silver nanoparticles. For the synthesis of nanoparticles, a 0.001 M aqueous solution of Silver Nitrate AgNO 3 (Sigma-Aldrich) and an aqueous solution of C. procumbens extract were used. With a volume-to-volume ratio of 1:1, that is, 5 mL of aqueous extract of C. procumbens as reducing agent and 5 mL of silver nitrate precursor salt as oxidizing agent. The silver ions were reduced to metallic silver within 6 h, showing a color change from light to dark brown.
Characterization of AgNPs. UV-Vis spectroscopy. UV-Vis spectroscopic analysis was performed to monitor the formation of AgNPs using a UV-Vis spectrophotometer (VE-5100UV spectrophotometer, USA) 28 . UV-Vis adsorption spectra were measured in a 1 cm quartz cuvette using 2 mL of the synthesized AgNPs solution. The samples were measured in wavelength ranges between 350 and 750 nm.
Catalytic degradation of congo red and malachite green dye. An aqueous solution of the malachite green and congo red dyes (10 -3 M) was prepared. Then 0.1 ml of AgNPs (0.941 µg/mL) solution was added to 2 mL of the dye solution. The dye degradation experiments were carried out under shaking and irradiation of a solar simulator (ScienceTech SF150B). The degradation of the solution was followed by measuring the absorption band characteristic of dye, using a UV-Vis spectrophotometer (VE-5100UV spectrophotometer, USA). To obtain the percentage of degradation: the dyes and the AgNPs were kept in linear agitation during the degradation period, additionally Eq. (1) was used.
where A 0 is the initial absorbance of the dye and A t is the absorbance of the dye at a specific time.
FTIR analysis. FTIR spectrums of the biogenic AgNPs and plant extracts were obtained in a Perkin Elmer spectrophotometer (L16000300 Spectrum Two LiTa, Llantrisant, UK), using the potassium bromide (KBr) pellet method. The samples were measured in wavelength ranging between 500 and 4000 cm −1 .
TEM analysis. Morphology and size distribution of AgNPs were investigated using a JEOL-2100 High-Resolution Transmission Electron Microscope (HR-TEM). Samples were prepared by placing a drop of AgNPs, dispersed in solution, followed by solvent evaporation.
Characterization of the size and zeta potential. The hydrodynamic mean diameter of the AgNPs was determined by photon correlation spectroscopy (PCS), using a 4700 C light-scattering device (Malvern Instruments, London, UK) working with a He-Ne laser (10 mW). The diffusion coefficient measured by the dynamic light scattering was used to calculate the size of the AgNPs by means of the Stokes-Einstein equation. The homogeneity of the size distribution is expressed as polydispersity index (PDI), which was calculated from the analysis of the intensity autocorrelation function (Zeta-Sizer Nano Z, Malvern Instruments, UK).

Broth dilution test.
Experiments on antimicrobial or antifungal activity were performed as described by . The cells (5 × 10 3 cells) were seeded in 96-well plates at 37 °C in a 5% CO 2 atmosphere and 24 h later, the cells were treated with different concentrations of biosynthesized AgNPs and incubated for 3 days. Then, the medium was removed and the cells were washed with PBS. Subsequently, 100 μL of 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazoyl bromide (MTT) (at a concentration of 0.2 mg/mL) was added to each well and incubated for three hours. After this time, the MTT reagent was removed, and the formazan crystals formed were dissolved by adding 100 μL of dimethyl sulfoxide (DMSO) per well and analyzed at 570 nm in a multi-well ELISA plate reader. The inhibitory concentration 50 (IC 50 ) was calculated with the GraphPad Prism program (GraphPad 6 Software San Diego, CA, EE. UU). All the experiments were plated in triplicate and were carried out at least twice. In addition, non-treated cells were used as controls. The therapeutic index was calculated for both the extracts and the AgNPs, from Eq. (2). The therapeutic index is expressed numerically as a ratio between the dose of the drug that causes death (lethal dose or DL) or a deleterious effect in a proportion "x" of the sample and the dose that causes the desired therapeutic effect (effective dose or DE) in the same or greater proportion "y" of the sample.
Statistic analysis. The data collection from the different biological studies represents the mean ± standard deviation. The two-tailed Student's t-test was used to compare the differences between two groups. A two-tailed p value < 0.05 is considered statistically significant.

Conclusions
Biogenerating silver nanoparticles from natural products such as the aqueous extract of C. procumbens is an environmentally friendly method, does not produce unwanted contaminants and is very easy to reproduce. Our results show that biogenerated AgNPs has potential as a microbial agent, anticancer agent, and also opens the possibility for the degradation of specific dyes. It is a simple procedure with several advantages such as costeffectiveness, biocompatibility for medical applications, as well as large-scale commercial production. These results give us an opening to continue investigating the applications that we will surely be reporting in future works.

Data availability
All data generated or analysed during this study are included in this published article. (2) TI = DL50 DE50